The use of cotransformation to introduce foreign DNA into eucaryotic cells has been described. PCT International Publication Numbers WO81/02425 and WO81/02426, published Sept. 3, 1981, and scientific articles identified therein. Gene amplification as a means for obtaining enhanced gene expression has also been described. Alt, F. W., et al., (1978). J. Biol. Chem. 253, 1357-1370, and Wahl, G. M., et al., (1979). J. Biol. Chem. 254, 8678-8689. Eucaryotic gene promoters are known. McKnight, S. L. et al., (1981). Cell 25, 385-398. Finally, cloned amplifiable genes have been described. Lee, D. E., et al., (1981). Nature 294, 228-232.
Known, cloned amplifiable genes whose amplification can be selected for include those whose product either (a) directly or indirectly interacts with an inhibitor of cell growth so as to render the inhibitor ineffective, or (b) is necessary for cell survival and can be inhibited by exogenously supplied substances. In both instances, the nature of the amplification process is such that increasing amounts of gene product must be produced in the presence of increasing amounts of inhibitor in order for cells to survive.
Gene products of type (b) include dihydrofolate reductase (DHFR) and aspartate transcarbamoylase (ATCase), which are amplified in cells resistant to high concentrations of methotrexate and PALA, respectively. The ATCase gene has been cloned. However, it has not been transformed into animal cells and shown to amplify. The intact DHFR gene has not been cloned. Expressing cDNAs of DHFR have been constructed and cloned, but have not been shown to amplify. Gene products of type (a) include the cloned, bacterial neomycin resistance gene whose expression in eucaryotes has been demonstrated. However, amplification of this gene has not been demonstrated.
The present invention discloses an unexpected, alternative route to amplification. Specific mutations can be introduced into genes coding for selectable or identifiable biochemical markers so as to render the genes functionally deficient. Following transformation, rare variant cell subclones can be identified which greatly amplify the expression of the mutant gene so that expression occurs at levels characteristic of the normal gene.
Specifically, a system involving gene transfer has been developed to study the nature of genetic rearrangements which are responsible for activation of mutant genes in mammalian cells. Cotransformation has been used to introduce a single copy of a plasmid containing an intact adenine phosphoribosyltransferase (aprt) gene along with a truncated thymidine kinase (tk) gene lacking its promoter sequence, into aprt.sup.- tk.sup.- mouse cells. Cells containing this plasmid transform to the aprt.sup.+ phenotype but remain tk.sup.-. Rare variants which mutate to the tk.sup.+ phenotype were considered likely to reveal some form of DNA rearrangement. All tk.sup.+ variants analyzed result from dramatic amplification of the promoterless tk gene along with significant lengths of flanking sequences. No other source of rearrangements are observed in over 40 tk.sup.+ mutants examined.
One striking feature of these amplified aprt.sup.+ tk.sup.+ clones is the rate at which aprt.sup.- tk.sup.+ variants appear. Examination of the structure of the amplified DNA indicates that all amplified units within a given aprt.sup.- cell bear an identical mutation. These data suggest that a correction procession is occurring at high frequency which may be designed to maintain sequence homogeneity within repeated genetic elements.